Summary of Work: We are examining the relationship between the X ray crystal structures of DNA polymerases and their fidelity. This year we focused on five efforts. 1. To test the hypothesis that HIV-1 RT fidelity depends on geometric selection at the active site, we studied the properties of an HIV-1 RT mutant containing an alanine in place of an arginine (Arg72) that forms part of the binding pocket for the nascent base pair. This R72A mutant RT has higher than normal average replication fidelity yet paradoxically places specific sequences at very high risk of mutation. 2. To test how HIV-1 RT interacts with the DNA minor groove upstream of the active site, we examined the ability of wild-type and mutant HIV-1 RTs to bypass bulky DNA adducts in the major groove that cause changes in minor groove dimensions. The results are consistent with the idea that hydrophobic interactions site of W266 in RTs minor groove binding track that occur 3 to 5 base pairs upstream of the active are critical determinants of DNA binding affinity and processivity. 3. We studied pol ? mutants in which an arginine that interacts with the minor groove at the active site is replaced by alanine or lysine. The results suggest that unfavorable interactions between an active site amino acid side chain and mispair-specific atoms in the minor groove contribute to DNA polymerase base substitution and frameshift error rates and error specificity. 4. DNA polymerase h reduces the incidence of sunlight-induced skin cancer in humans by >1000-fold. In FY?00 we provided evidence that pol h copies undamaged DNA with much lower fidelity than any other template-dependent DNA polymerase studied to date. Its extraordinarily low fidelity indicates a relaxed requirement for correct base pairing geometry and suggests that the function of pol h may be tightly controlled to prevent potentially mutagenic DNA synthesis in human cells. 5. To test this hypothesis that some of the instability of triplet repeat sequences associated with hereditary human diseases may result from strand slippage during DNA synthesis, we studied the fidelity of replication of CAG repeats. The results support the slippage hypothesis and the idea that slipped intermediates are inefficiently corrected by exonucleolytic proofreading.